The c-Myc transcription factor is a potent inducer of cell proliferation and transformation, and elevated levels of c-Myc protein are observed in most human tumors. However, c-Myc overexpression also induces apoptosis, which limits c-Myc's tumorigenic potential. There are two conserved phosphorylation sites, Threonine 58 (T58) and Serine 62 (S62) that differentially regulate c-Myc protein stability in response to mitogenic stimulation, where S62 phosphorylation increases c-Myc stability, while T58 phosphorylation decreases c-Myc stability. Recent evidence suggests that phosphorylation at these sites also regulates c-Myc's apoptotic versus tumorigenic potential. Specifically, low phosphorylation at T58 and high phosphorylation at S62, which is the signature for more stable c-Myc, appears to suppress c-Myc's apoptotic activity and enhance its proliferative properties. Recent results indicate that lower T58 and higher S62 phosphorylation occurs in some tested human cancer cell lines with aberrantly stabilized c-Myc protein due to deregulation of the pathway that controls c-Myc degradation. Moreover, similarly altered phosphorylation of c-Myc has been detected in primary breast cancer samples. This suggests that cancer cells may contain a more oncogenic form of c-Myc. The objective of this grant is to examine the role of phosphorylation at T58 and S62 in controlling c-Myc's apoptotic versus proliferative activity, and whether this mechanism contributes to c-Myc's transforming activity in human cancer. The following specific aims will be pursued: 1) Examine the activity of c-Myc T58 and S62 phosphorylation mutants in vivo using a unique mouse model; 2) Investigate mechanisms that could underlie the different phenotypic responses to expression of c-Myc T58 and S62 phosphorylation mutants; and 3) Examine the phosphorylation status of c-Myc at T58 and S62 in human cancer cells and whether manipulation of c-MycWT phosphorylation can affect its oncogenic potential. These aims involve the study of novel inducible c-myc knock-in mice that express either wild-type c-Myc or c-Myc T58 or S62 phosphorylation mutants in specific tissues, in vitro and in vivo assays to investigate the underlying mechanisms of how these sites affect c-Myc activity, and an analysis of human cancer to explore the relevance of altered phosphorylation at these sites and whether cell transformation can be affected by non-genetic manipulation of c-Myc T58 and S62 phosphorylation. This research has important therapeutic implications, since targeting the pathway that regulates T58 and S62 phosphorylation could potentially affect both c-Myc expression levels and tumorigenic activity. PUBLIC HEALTH RELEVANCE: The proposed research focuses on understanding how the oncogenic potential of the transcription factor c-Myc is affected by its phosphorylation status at two highly conserved sites, which also regulate its protein stability. Importantly, elevated expression of c-Myc is widely observed in many different human tumors and therefore understanding mechanisms that increase or decrease c-Myc's oncogenic potential are critical to the development of future therapies targeting this potent oncoprotein.